US 20070009379 A1
Described herein are novel devices for the study of transport characteristics of complex or simple fluids, interactions among molecules in suspension, interactions between molecules in suspension and wall-bound molecules, and biochemical sensing devices made of reservoirs for fluid containment linked by a nanotubes. Also disclosed are methods of delivering medicaments and monitoring fluidic interactions of molecules or analytes.
1. An analytical device comprising:
a barrier structure defining two reservoirs for fluid containment; and
at least one nanotube between said reservoirs, the lumen of which nanotube is at least partially observable by electron or ion-beam microscopy, the openings of said nanotube being in fluid communication with each of said reservoirs.
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14. An analytical device comprising:
a barrier structure defining at least two reservoirs for fluid containment;
at least one nanotube between said reservoirs and the ends of said nanotubes are in fluid communication with said reservoirs.
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31. A probe comprising:
a barrier structure defining one reservoir for fluid containment; and
a nanotube having an opening proximal to and in fluid communication with said reservoir and an opening distal to said reservoir for insertion.
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44. An array comprising more than one probe comprising:
barrier structures on said substrate defining reservoirs for fluid containment; and
more than one nanotube having an opening proximal to and in fluid communication with said reservoir and an opening distal to said reservoir for insertion.
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60. A method of delivery comprising:
placing a medicament in a fluid;
placing said fluid into at least one probe comprising:
a barrier structure on said substrate defining a reservoir for fluid containment; and
a nanotube having an opening proximal to and in fluid communication with said reservoir and an opening distal to said reservoir; and
delivering said medicament contained within said fluid into said biological membrane through said nanotube.
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74. A method of analysis comprising:
placing an analyte in a fluid;
placing said fluid into an array comprising more than one probe comprising:
a barrier structure on said substrate defining reservoirs for fluid containment;
a nanotube having openings proximal to and in fluid communication with said reservoirs and opening distal to said reservoirs; and
monitoring the interaction of said analyte within said nanotubes.
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86. A method of monitoring the fluidic interactions of analytes comprising:
placing said analytes in a fluid;
placing said fluid into at least one device comprising:
a barrier structure defining two reservoirs for fluid containment; and
a nanotube between said reservoirs and the ends of which nanotube are in fluid communication with said reservoirs; and
observing the fluidic interactions of said analytes within said nanotube.
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The present invention relates to nanotube based sensors and probes used for biochemical and chemical sensing and processing and for electron and optical microscopy of chemical and biological interactions in liquids and gases. Also disclosed are methods of medicament transportation and monitoring molecular interactions.
One of the obstacles encountered when studying nano-scale phenomena is the limited resolution of visible light. When dealing with solid or frozen materials, one can take advantage of the relatively small wavelengths of electrons to visualize phenomena at sub-nanometer length scales. Unfortunately, however, conventional electron microscopy requires vacuum conditions, and, in the past, this has precluded its use for the study of volatile fluids. With present day technology, at best, one can operate with environmental chambers that allow the introduction of humid gases. The conditions prevailing in the environmental chamber are very different from the ones experienced, for example, by biological molecules in aqueous solutions and catalytic reactions in general. This limits one's ability to carry out studies of biological interactions and chemical reactions under controlled conditions.
It is envisioned that experiments currently conducted with near field microscopy and total internal reflection (TIRF) microscopy can be duplicated with electron microscopy with much higher resolution than is currently feasible. The molecules to be observed can be tagged with particles, atoms, and possibly observed directly without a label. As a result, various reactions and interactions in liquid and gaseous environments may be studied.
A common method for the detection of particles and biological substances is to transmit the analyte through a small tube or pore and monitor the effect of the presence of the analyte on the ionic current. The presence of an analyte suppresses or blocks the ionic current. The magnitude of the blockage and its duration can be used to characterize the size of the analyte. To make the detection process specific, one can use functionalized carriers such as particles or known molecules that bind the analyte specifically and monitor the effect of the analyte on the characteristics of the carrier. The sensitivity of this biosensing technique depends on the size of the pore or tube. The small diameter of the nanotubes would facilitate the construction of high sensitivity devices.
Currently, pulled glass micropipettes are used to study the properties of cells and to exchange material with a cell's interior. These techniques are intrusive and typically limited to operating with a single cell at a time. There is a need for less intrusive probes.
Currently scanning probes allow one to probe the mechanical and electrical properties of samples. There is an unmet need for probes that can exchange fluids and molecules with the scanned sample.
The present invention provides analytical devices comprising a substrate, a barrier structure defining two reservoirs for fluid containment, and at least one nanotube between the reservoirs, the lumen of which nanotube is at least partially observable by electron, optical, or ion beam microscopy, and the openings of which nanotube are in fluid communication with said reservoirs.
The techniques described herein facilitate the fabrication of devices comprising a plurality of nanotubes similarly or differently sized. The nanotubes may also be similarly or differently functionalized to interact with the same or different reservoirs. Groups of nanotubes may communicate with shared reservoirs or with individual reservoirs.
The present invention also includes methods of monitoring the fluidic interactions of molecules. The method comprises placing the molecules in a fluid, placing the fluid into a device of the present invention, causing the fluid to flow from one reservoir to another reservoir through the nanotube, and observing the fluidic interaction of the molecules within the nanotube.
Also disclosed are cellular or scanning probes comprising a substrate, a barrier structure on the substrate defining a reservoir for fluid containment, and a nanotube. The nanotube has an opening proximal to and in fluid communication with the reservoir and the distal opening of the nanotube is exposed. There are also embodiments in which the distal opening is used for insertion into a biological membrane. Such embodiments may facilitate the introduction into or withdrawal from a cell or molecule in fluids.
There are also methods of delivery for a medicament comprising placing a medicament in a fluid, then placing that fluid into at least one probe or device of the present invention, and then delivering the medicament contained within the fluid into a biological membrane through the nanotube.
Arrays comprising more than one cellular or scanning probe are disclosed. The arrays comprise a substrate and barrier structures on said substrate defining reservoirs for fluid containment. They also comprise nanotubes having openings proximal to and in fluid communication with the reservoirs and openings distal to the reservoirs for insertion into a biological membrane. More than one probe may be located on the same substrate.
The fabrication techniques described herein facilitate the fabrication of devices that allow a plurality of probes of different sizes and functionalization to interact with a single or group of cells. Groups of probes may communicate with shared reservoirs or with individual reservoirs.
Disclosed herein are hybrid methods for the fabrication of nanotube-based fluidic devices, devices for biochemical sensing and processing, and devices that facilitate electron microscopy of biological and chemical interactions in liquids or pressurized gases. The embodiments of the present invention allow for the transport of simple and complex fluids from one reservoir to the other or to a biological membrane through a nanotube. The transportation may be facilitated by means of an electric field across the electrodes, such as by electroosmosis or electrophoresis, by electro-wetting and electro-migration and by diffusion. The fluids may also be made to flow by surface tension or pressure. Molecules may be transmitted by the directed motion of cargo carrying, processive molecular motors. The activity inside the nanotube may be observed through a pathway in the substrate and between the reservoirs to the lumen of the nanotube. The contents of the nanotube may be observed, for example, with optical, fluorescent, and electron microscopes or ion-beam microscopes. Alternatively, they can be measured with electrical means such as the monitoring of the ionic current through the liquid confined inside the nanotube and the monitoring of the effect of the contents on the nanotube wall's electrical, optical, and mechanical properties.
One embodiment that may be preferred provides devices comprising a substrate; a barrier structure defining two reservoirs for fluid containment; and a nanotube between the reservoirs. The openings of the nanotube are in fluid communication with the reservoirs. The term “reservoirs,” as used herein, is considered a chamber and its interior used for storing fluid or a conduit that facilitates the supply of fluids. The nanotube connects the two reservoirs and facilitates the transport of fluids from one reservoir to the other. Such an embodiment may be utilized as a biosensor for the detection and characterization of molecules, as a device to study the transport characteristics of simple and complex confined liquids. Some embodiments may be used as a miniature containment vessel allowing for electron microscopy of reactions and interactions in liquids and pressurized gases within the vacuum environment of the electron microscope.
For some embodiments, additional steps may be incorporated to cap the reservoirs 110 with a cover layer structure 240 as seen in
In other embodiments, the biochemical sensor may further comprise a pathway 200 through said polymer and between said reservoirs 110 to the lumen of said nanotube 100. This pathway allows for the contents of the nanotube 100 to be observed using microscopy known in the art.
The embodiment depicted in
Additional steps may be incorporated to add a cover structure 240. Two approaches are described. A cover structure 240, formed of a glass slide with pre-opened windows 250 and spun on one side of the structure with a thin layer of adhesive film may be used to make the embodiment as shown in step 9. The cover structure 240 structure may also be introduced at step 7 prior to opening of the reservoirs using standard microfabrication processes. Inlet and outlet ports may be sealed off with properly engineered closures to make the devices vacuum tight.
In addition to the applications listed above, there are embodiments used as scanning or cellular probes to penetrate biological membranes to exchange material, deliver material, or extract material with minimal intrusion and high resolution and serve as a nanoelectrode.
The exposed opening of the nanotube 100 that is distal to the reservoir is for insertion into a biological membrane. Once inserted, a sample may be retrieved from the membrane so that it may be observed or analyzed within the nanotube 100 or reservoir 110. In other embodiments, once the distal opening of the nanotube 100 is inserted, the contents of the reservoir 110 may be injected into the membrane. The scanning probes may be functionalized to facilitate selective binding and transport. There are embodiments that may also serve as nanoelectrodes.
Multiple probes may make an array. The probes may communicate, in parallel, with multiple cells for massive parallel processing. Such an embodiment may be seen in
The cellular probes of the present invention may be smaller or have a higher aspect ratio (i.e. the ratio of the tip's length to its width) than probes currently used. There may be a plurality of probes with individual nanotubes or groups of nanotubes communicating with individual cells. Some embodiments may comprise nanotubes connected to microfluidic conduits for continuous supply of reagents.
Some embodiments of the probes of the present invention may be constructed according to process steps 1-7 illustrated in
Alternatively, devices may be fabricated with the two photon lithography as shown in
An embodiment similar to the one depicted in
It will be appreciated that the devices and probes of the present invention lend themselves to certain novel methods. To that end, there are methods of delivering a medicament comprise placing a medicament in a fluid, placing the fluid into at least one probe as described herein, and delivering the medicament contained within the fluid into a biological membrane through the nanotube. The biological membrane may be a cell membrane. The medicament to be delivered may comprise protein, hormones, antibiotics, enzymes, or chemical agents. In some embodiments, there is more than one probe being utilized. In such embodiments, the medicament contained within the fluid may be injected into a membrane through more than one probe. Also, the probes in such embodiments may act in parallel during injection.
There are also methods of monitoring the fluidic interactions of an analyte or molecule or between molecules in suspension and molecules immobilized to the nanotube's wall comprise placing the analytes or molecules in a fluid, placing the fluid into a device as described in the present disclosure, and observing the fluidic interactions of said analyte within the nanotube. There are also embodiments for monitoring the interactions of an analyte wherein the fluid is placed into an array comprising more than one probe and more than one probe is located on the same substrate. The diameters of the nanotubes in such embodiments may be the same or different. The functionalization of each nanotube may also be the same or different in the probes in array embodiments. In some embodiments, the methods include a step where the fluid is caused to flow from one reservoir to another reservoir or through a membrane. The flow may be caused by electroosmosis or electrophoresis.
Ionic current measurements through embodiments of the present invention have been carried out, and the present invention has utility as a highly sensitive Coulter counter. The experimental observations of the ion transmission and the particle translocation provide evidence that the hybrid fabrication methods of some embodiments provide a well-functioning nanotube based-fluidic device that can be used as a high sensitivity particle counter. Furthermore, the disclosed devices and methods should allow one to position multiple nanotubes with different diameters on the same substrate facilitating massive parallel processing, and extending the technique to nanotubes of molecular dimensions should also be feasible.
The observing steps may be performed via electron microscopy. Observation may also be done using optical or fluorescent microscopy. For optical microscopy, the nanotube confines a minute quantity of labeled analytes that can be observed with minimal interference. The fluidic interactions that may be observed comprise the effects of the analyte or molecule on an ionic current, the size and velocity of the analyte or molecule. Also, the resonance frequency, the electrical resistance, or impedence of the nanotube may be observed. The analyte or molecule may also comprise a drug to be screened or tested. A vacuum may also be applied to the device in some method embodiments.
The nanotubes used in the embodiments of the present invention may be amorphous, multi-walled, or single walled. The physical properties of the nanotube may be modified by chemical or thermal treatment. The walls of the nanotubes may be unmodified or functionalized with ligands or immobilized ligands. The nanotubes may also be electrically charged. A plurality of nanotubes with similar or different diameters and functionalization may be integrated into some embodiments. Groups of nanotubes may communicate with a single reservoir or each nanotube may communicate with an individual reservoir.
Analytes or molecules transmitted within the nanotube may also affect a nanotube's properties. As a result, the mechanical, optical, or electrical properties of the nanotube may be observed in some methods of the present invention. These properties include mass, stiffness, elastic properties, and electric properties. A nanotube's inner wall may also be functionalized to selectively bind or adsorb specific target analytes or molecules. Since the nanotube wall thickness is small, the presence of target analytes within the nanotube and the binding of analytes to the nanotube walls can be sensed by monitoring the wall's mechanical properties such as the natural frequency of the nanotube vibrations or the electrical properties such a electrical resistance and impedance. Adsorbed analytes or molecules may also affect the nanotube wall's optical properties and the electroosmotic velocity of fluids inside the nanotube. The interactions between molecule in suspension and those attached to the nanotube wall may also be observed.
Methods of making some embodiments of the disclosed devices are depicted in
To demonstrate that the nanotube device can be used to transport ions in aqueous solutions, the nanotubes were filled with a 0.1 M KCl electrolyte solution and measured the current-voltage characteristics. Shown in the
The I-V curve of the nanotube device (
Experimental data demonstrates both the transport of fluid inside a carbon nanotube and the feasibility of observing the liquid motion. The motion of the fluid in